The present invention relates to a novel method of chemical preparation of xcex2-D-glucopyranosyl-(1xe2x86x923)-D-glucopyranose of formula (I), commonly called laminaribiose. 
Laminaribiose is a disaccharide which is used notably in the agricultural field and as an antiseptic.
This disaccharide is in general obtained by hydrolysis or by acetolysis of natural polysaccharides of plant origin (see Villa, Phaff, Notario, Carbohydr. Res., 1979, 74, 369; Kusama, Kusakabe, Zama, Murakami, Yasui, Agric. Biol. Chem., 1984, 48, 1433; Wang, Sakairi, Kuzuhara, Carbohydr. Res., 1991, 219, 133; Moreau, Viladot, Samain, Planas, Driguez, Bioorg. Med. Chem., 1996, 4, 1849).
Laminaribiose can also be prepared chemically, notably by methods derived from the Koenigs-Knorr method of O-glycosylation (see Koenigs, Knorr, Ber. Dtsch. Chem. Ges., 1901, 34, 957) which makes use of glycosyl halides as glycosyl donors.
A first method has thus been proposed by Freudenberg and von Oertzen in 1951 (see Freudenberg, von Oertzen, Justus Liebigs Ann. Chem., 1951, 574, 37), and a second method has been described by Bxc3xa4chli and Percival in 1952 (see Bxc3xa4ichli, Percival, J. Chem. Soc., 1952, 1243).
The major drawbacks of these two methods reside in a purification which is difficult to carry out and in an overall yield which is lower that 10%.
A third method has been proposed by Takeo in 1979 (see Takeo, Carbohydr. Res., 1979, 77, 245), but it necessitates several steps of selective protection and deprotection of the hydroxyls of the acceptor used which is in glucopyranose form.
It has also been proposed to form laminaribiose from ortho-esters (see Kochetkov, Bochtov, Sokolovskaya, Snyatkova, Carbohydr. Res., 1971, 16, 17). This method does however prove to be difficult to carry out and enables laminaribiose to be obtained only with an overall yield neighbouring 10%.
Under these circumstances, the aim of the present invention is to provide a novel method of chemical preparation of laminaribiose which has a limited number of steps which are easy to carry out and which enables the product sought after to be obtained in pure form with a high overall yield.
The solution in accordance with the present invention to solve this technical problem consists of a method of preparing laminaribiose comprising a step of glycosidic coupling between a glycosyl donor and a glycosyl acceptor, characterised in that:
the glycosyl donor is in pyranose form and is of formula (II): 
xe2x80x83in which:
R1 represents:
an alkyl or haloalkyl radical having 1 to 6 carbon atoms
an aryl radical which is non-substituted or substituted with one or more groups selected from a halogen atom, an alkoxy radical having 1 to 6 carbon atoms or a nitro group;
X represents an electrophilic leaving group selected from:
a group of formula S(O)nRxe2x80x2 in which Rxe2x80x2 represents an alkyl radical having 1 to 6 carbon atoms, an aryl radical which is non-substituted or substituted with an alkoxy group having 1 to 6 carbon atoms, a nitro or acetamide group, and n is an integer equal to 0 or 1; or
a trichloroacetimidate group;
the glycosyl acceptor is in furanose form and is of formula (III) 
xe2x80x83in which:
R2 and R3 together form a methylidyl, ethylidyl, trichloroethylidyl, isopropylidyl, hexafluoroisopropylidyl, cyclopentylidyl, cyclohexylidyl, cycloheptylidyl, butylidyl, 1-tert-butylethylidyl, 1-phenylethylidyl, benzylidyl, methoxybenzylidyl, or 1-phenylbenzylidyl radical ; and
R4 and R5 together form a methylidyl, ethylidyl, trichloroethylidyl, isopropylidyl, hexafluoroisopropylidyl, cyclopentylidyl, cyclohexylidyl, cycloheptylidyl, butylidyl, 1-tert-butylethylidyl, 1-phenylethylidyl, benzylidyl, methoxybenzylidyl, or 1-phenylbenzylidyl radical, or independently represent a benzyl, acetyl, benzoyl, chlorobenzoyl, methoxybenzoyl, nitrobenzoyl, allyl, chlorobenzyl, methoxybenzyl or nitrobenzyl radical;
said coupling step is carried out in solution in an anhydrous organic solvent, at a temperature between xe2x88x9280xc2x0 C. and 40xc2x0 C., for a period of 1 minute to 8 hours, in the presence of a suitable promoter selected from:
a source of halonium ions, combined or not with a Lewis acid or a salt of a strong acid, in the case in which X represents an S(O)nRxe2x80x2 group as defined above in which n is equal to 0;
a Lewis acid combined with an amine, in the case in which X represents an S(O)nRxe2x80x2 group as defined above in which n is equal to 1; or
a Bronsted acid or a Lewis acid, in the case in which X represents a trichloroacetimidate group ; and
the reaction product thus obtained, neutralised and purified, being subjected to a deprotection treatment to give, after purification, laminaribiose.
It has been discovered, and this constitutes the basis of the present invention, that it was possible to chemically prepare laminaribiose with a limited number of steps which enable a relatively high overall yield to be obtained, by a judicial choice of the glycosyl donor and of the glycosyl acceptor, as well as of the promoter used during the coupling reaction.
In the description and claims:
 less than  less than alkyl radical having 1 to 6 carbon atoms greater than  greater than  is understood as meaning any linear or branched hydrocarbon chain, such as a methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, hexyl, or isohexyl radical, for example;
 less than  less than haloalkyl radical having 1 to 6 carbon atoms greater than  greater than  is understood as meaning any alkyl radical 1 to 7 hydrogen atoms of which are substituted by 1 to 7 halogen atoms, such as a chloromethyl radical, a bromomethyl radical, a trifluoromethyl radical, a 2,2,2-trifluoroethyl radical, a pentafluoroethyl radical, or a heptafluoropropyl radical, for example;
 less than  less than aryl radical greater than  greater than  is understood as meaning an aromatic ring having 5 or 6 carbon atoms or heteroatoms, such as a phenyl, pyridyl, thienyl, furanyl, or pyrimidyl radical, for example.
The glycosyl donor of formula (II) mentioned above as well as the glycosyl acceptor of formula (III) mentioned above can be obtained relatively easily, in one or two steps, from D-glucose.
Advantageously, the glycosyl donor will in general be selected from the compounds of formula (II) mentioned above in which:
R1 represents a radical selected from the group consisting of methyl, chloromethyl, trifluoromethyl, tert-butyl, phenyl, chlorophenyl, methoxyphenyl and nitrophenyl radicals;
X represents a radical selected from the group consisting of thiomethyl, thioethyl, thiopropyl, thiophenyl, thionitrophenyl, and thiopyridyl radicals.
In general, the promoter used during the coupling step mentioned above will be selected from:
N-bromosuccinimide or N-iodosuccinimide, combined or not with a Lewis acid selected from ferric chloride, copper ditriflate, tin ditriflate, boron trifluoride dietherate, tin or zirconium tetrachloride, methyl triflate, trimethyl- (or triethyl-) silyl triflate, silver triflate, cadmium ditriflate, cobalt ditriflate, nickel ditriflate, zinc ditriflate, bismuth tritriflate, iron tritriflate, gallium tritriflate, or with a salt of a strong acid such as tetrabutylammonium triflate, in the case in which X represents an S(O)nRxe2x80x2 group as defined above in which n is equal to O,
a Lewis acid selected from triflic anhydride, ferric chloride, copper ditriflate, tin ditriflate, boron trifluoride dietherate, tin or zirconium tetrachloride, methyl triflate, trimethyl- (or triethyl-) silyl triflate, silver triflate, cadmium ditriflate, cobalt ditriflate, nickel ditriflate, zinc ditriflate, bismuth tritriflate, iron tritriflate, gallium tritriflate, combined with an amine particularly such as di-tert-butylmethylpyridine, in the case in which X represents an S(O)nRxe2x80x2 group as defined above in which n is equal to 1, and
a Bronsted acid particularly such as triflic acid or para-toluenesulphonic acid or a Lewis acid selected from triflic anhydride, ferric chloride, copper ditriflate, tin ditriflate, boron trifluoride dietherate, tin or zirconium tetrachloride, methyl triflate, trimethyl- (or triethyl-) silyl triflate, silver triflate, cadmium ditriflate, cobalt ditriflate, nickel ditriflate, zinc ditriflate, bismuth tritriflate, iron tritriflate, gallium tritriflate, in the case in which X represents a trichloroacetimidate group.
In a currently preferred embodiment of the method according to the invention:
the glycosyl donor is of formula (II) mentioned above in which:
R1 represents a phenyl radical; and
X represents an S(O)nRxe2x80x2 radical in which n is equal to O and Rxe2x80x2 represents an ethyl or phenyl radical;
the glycosyl acceptor is of formula (III) mentioned above in which:
R2, R3 and R4, R5 together form a cyclohexylidyl or isopropylidyl radical.
In this particular embodiment, the promoter used during the coupling reaction is constituted of a mixture of N-iodosuccinimide and of tin ditriflate, preferably in proportions between 1:0.5 and 1:0.005.
In general, the coupling step mentioned above is carried out in solution in dichloromethane, 1,2-dichloroethane or toluene, preferably in the presence of molecular sieves, at a temperature between xe2x88x9230xc2x0 C. and 30xc2x0 C., for a period of 1 minute to 6 hours, preferably at 10xc2x0 C. for 30 minutes.
It will be possible for the respective amounts of glycosyl donor, of glycosyl acceptor and of promoter to be determined easily by the person skilled in the art.
In general, the coupling reaction can be carried out by allowing to react:
one equivalent of glycosyl acceptor;
one to two equivalents of glycosyl donor; and
one to two equivalents of promoter;
in 5 to 200 equivalents by weight, with respect to the acceptor, of a solvent.
Advantageously, an organic solvent such as dichloromethane, 1,2-dichloroethane or toluene will be used, in the presence of molecular sieves (intended for trapping the acid which can form during the reaction) such as 4 xc3x85 molecular sieves for example, used in an amount of 10 to 200 mg/ml of solvent.
The product obtained by the coupling reaction mentioned above is generally neutralised and then purified.
The neutralisation can be carried out by adding an organic base, preferably triethylamine or ethanolamine, or even by adding an inorganic base, preferably sodium or potassium carbonate or hydrogen carbonate, followed by filtering the salt obtained.
The purification can be carried out:
either by chromatography, for example on a silica gel or active charcoal column,
or by fractional crystallisation preferably in an organic solvent or a mixture of organic solvents such as ethyl ether, ethyl acetate, cyclohexane or ethanol.
The product of the coupling reaction, neutralised and purified leads, via a deprotection treatment followed by a purification, to laminaribiose.
In general, the deprotection treatment mentioned above comprises two steps, the first consisting of a partial deprotection of the product of the coupling reaction, by cleavage of the acetal groups originating from the glycosyl acceptor.
Within the context of the method in accordance with the present invention, the deprotection treatment comprises:
a) cleaving the acetal groups originating from the glycosyl acceptor by an acidic treatment in an aqueous or hydro-organic medium, or in the presence of an acidic resin;
b) purifying the product thus obtained;
c) transesterifying or hydrolysing the product obtained in step b); and
d) purifying the product thus obtained.
The cleavage reaction a) mentioned above will preferably be carried out in an acidic hydro-organic medium, such as in a mixture of equal volumes of trifluoroacetic acid and water for example, at a temperature between 10 and 70xc2x0 C. for a period of 1 hour to 10 days and in this case, the partially deprotected product obtained will be purified by fractional crystallisation, preferably in methanol, or by chromatography.
It is also possible for acetic acid, oxalic acid, formic acid, sulphuric acid, hydrochloric acid, and phosphoric acid, to be used instead of trifluoroacetic acid.
Within the context of the method in accordance with the present invention, the transesterification step c) mentioned above will be carried out in an alcoholic solvent such as methanol or ethanol in the presence of a catalytic amount of sodium or of sodium or potassium methoxide or ethoxide, for a period of 1 minute to 10 days.
The product of transesterification thus obtained will generally be purified by a method comprising:
d1) neutralising the product obtained in step c);
d2) removing the benzoic ester formed, either by azeotropic evaporation with water, or by extraction with an organic solvent;
d3) concentrating under reduced pressure the residual aqueous phase;
d4) optionally, lyophilising or crystallising the laminaribiose thus obtained in a hydro-alcoholic mixture.